Autoshortloop feedback regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by its metabolite, GnRH-(1-5).

Given the central role of the decapeptide gonadotropin-releasing hormone (GnRH) in reproductive function, our long-term objective is to delineate the underlying mechanism regulating these reproductive processes. The outcome of GnRH secretion is in part dependent on the proteolytic metabolism of this decapeptide. In contrast to the belief that the metabolism of GnRH serves only as a degradative process that removes excess GnRH, we have shown that a metabolite of the decapeptide, GnRH-(1-5), can directly regulate GnRH gene expression and reproductive behavior. To further characterize the effect of GnRH-(1-5) on GnRH neuronal function, we determined whether GnRH-(1-5) can directly regulate GnRH secretion and pulsatility using an in vitro perifusion system. We compared the effect of GnRH-(1-5) on GnRH secretion in the immortalized GnRH neuron (GT1-7 cell line), whole rat hypothalamic explant, and enzymatically dispersed rat hypothalamic cells. Tissue preparations were perifused continuously for 9 h during which a 3-h challenge with GnRH-(1-5) was administered (4-6 h). The results show that treatment with GnRH-(1-5) increased (p < 0.05) the mean GnRH secretion and the amplitude of the pulses but not the pulse frequency. The present study supports the notion that GnRH-(1-5) is functionally capable of regulating the reproductive neuroendocrine system.

Hypothalamic molecular changes underlying natural reproductive senescence in the female rat.

The role of the hypothalamus in female reproductive senescence is unclear. Here we identified novel molecular neuroendocrine changes during the natural progression from regular reproductive cycles to acyclicity in middle-aged female rats, comparable with the perimenopausal progression in women. Expression of 48 neuroendocrine genes was quantified within three hypothalamic regions: the anteroventral periventricular nucleus, the site of steroid positive feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback and pulsatile GnRH release; and the median eminence (ME), the site of GnRH secretion. Surprisingly, the majority of changes occurred in the ARC and ME, with few effects in anteroventral periventricular nucleus. The overall pattern was increased mRNA levels with chronological age and decreases with reproductive cycle status in middle-aged rats. Affected genes included transcription factors (Stat5b, Arnt, Ahr), sex steroid hormone receptors (Esr1, Esr2, Pgr, Ar), steroidogenic enzymes (Sts, Hsd17b8), growth factors (Igf1, Tgfa), and neuropeptides (Kiss1, Tac2, Gnrh1). Bionetwork analysis revealed region-specific correlations between genes and hormones. Immunohistochemical analyses of kisspeptin and estrogen receptor-α in the ARC demonstrated age-related decreases in kisspeptin cell numbers as well as kisspeptin-estrogen receptor-α dual-labeled cells. Taken together, these results identify unexpectedly strong roles for the ME and ARC during reproductive decline and highlight fundamental differences between middle-aged rats with regular cycles and all other groups. Our data provide evidence of decreased excitatory stimulation and altered hormone feedback with aging and suggest novel neuroendocrine pathways that warrant future study. Furthermore, these changes may impact other neuroendocrine systems that undergo functional declines with age.

Disruption of reproductive aging in female and male rats by gestational exposure to estrogenic endocrine disruptors.

Polychlorinated biphenyls (PCBs) are industrial contaminants and known endocrine-disrupting chemicals. Previous work has shown that gestational exposure to PCBs cause changes in reproductive neuroendocrine processes. Here we extended work farther down the life spectrum and tested the hypothesis that early life exposure to Aroclor 1221 (A1221), a mixture of primarily estrogenic PCBs, results in sexually dimorphic aging-associated alterations to reproductive parameters in rats, and gene expression changes in hypothalamic nuclei that regulate reproductive function. Pregnant Sprague Dawley rats were injected on gestational days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or estradiol benzoate (50 µg/kg). Developmental parameters, estrous cyclicity (females), and timing of reproductive senescence were monitored in the offspring through 9 months of age. Expression of 48 genes was measured in 3 hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC), and median eminence (females only) by real-time RT-PCR. Serum LH, testosterone, and estradiol were assayed in the same animals. In males, A1221 had no effects; however, prenatal estradiol benzoate increased serum estradiol, gene expression in the AVPV (1 gene), and ARC (2 genes) compared with controls. In females, estrous cycles were longer in the A1221-exposed females throughout the life cycle. Gene expression was not affected in the AVPV, but significant changes were caused by A1221 in the ARC and median eminence as a function of cycling status. Bionetwork analysis demonstrated fundamental differences in physiology and gene expression between cycling and acyclic females independent of treatment. Thus, gestational exposure to biologically relevant levels of estrogenic endocrine-disrupting chemicals has sexually dimorphic effects, with an altered transition to reproductive aging in female rats but relatively little effect in males.

Endocrine disruption of brain sexual differentiation by developmental PCB exposure.

In mammals, sexual differentiation of the hypothalamus occurs during prenatal and early postnatal development due in large part to sex differences in hormones. These early organizational processes are critically important for the attainment and maintenance of adult reproductive functions. We tested the hypothesis that perinatal exposure to polychlorinated biphenyls (PCBs) that disrupt hormonal pathways would perturb reproductive maturation and the sexually dimorphic development of neuroendocrine systems in the preoptic area (POA). Pregnant Sprague-Dawley rats were injected on gestational d 16 and 18 with vehicle (dimethylsulfoxide), Aroclor 1221 (A1221, an estrogenic PCB mix), a reconstituted PCB mixture representing those highest in human body burden (PCBs 138, 153, 180), or estradiol benzoate, an estrogenic control. Male and female pups were monitored for somatic and reproductive development. In adulthood, some rats were perfused and used for immunohistochemistry of estrogen receptor α, kisspeptin, and coexpression of Fos in GnRH neurons. Other rats were used to obtain fresh-frozen POA dissections for use in a PCR-based 48-gene expression array. Pubertal onset was advanced and estrous cyclicity irregular in endocrine-disrupted females. Furthermore, sexual differentiation of female neuroendocrine systems was masculinized/defeminized. Specifically, in the adult female anteroventral periventricular nucleus, estrogen receptor α-cell numbers and kisspeptin fiber density were significantly decreased, as was GnRH-Fos coexpression. PCR analysis identified androgen receptor, IGF-I, N-methyl-d-aspartate receptor subunit NR2b, and TGFß1 mRNAs as significantly down-regulated in endocrine-disrupted female POAs. These data suggest that developmental PCBs profoundly impair the sexual differentiation of the female hypothalamus.

Pulsatile gonadotropin-releasing hormone release from hypothalamic explants of male marmoset monkeys compared with male rats.

The present study was conducted to quantify in vitro gonadotropin-releasing hormone (GnRH) release parameters in the male marmoset. We established primary cultures of marmoset hypothalamic tissues for approximately 2 days (marmosets) to assess GnRH release profiles in vitro in hypothalamic explants from testis-intact and gonadectomized males. Pulsatile GnRH release profiles were readily demonstrated from in vitro hypothalamic explants isolated from adult male marmoset monkeys. Gonadectomy of male marmosets resulted in elevated mean GnRH and pulse amplitude from hypothalamic explants on the 1st day of culture (day 0). GnRH pulse amplitude increased by day 2 in approximately 67% of hypothalamic explants from testis-intact marmosets, suggesting release from an endogenous regulator of GnRH. We also measured GnRH release profiles in vitro in hypothalamic explants from testis-intact and gonadectomized rats. Male rats showed no changes in any concentration or frequency release parameters for GnRH following gonadectomy or during successive days in culture. The present study represents a unique examination of GnRH release from male marmoset monkey hypothalamic tissue and compares release dynamics directly with those obtained from male rat, suggesting a species difference in feedback regulation of GnRH release.

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153.

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.

Effects of perinatal polychlorinated biphenyls on adult female rat reproduction: development, reproductive physiology, and second generational effects.

Perinatal exposures to endocrine-disrupting chemicals, such as polychlorinated biphenyls (PCBs), can cause latent effects on reproductive function. Here, we tested whether PCBs administered during late pregnancy would compromise reproductive physiology in both the fetally exposed female offspring (F1 generation), as well as in their female offspring (F2 generation). Pregnant Sprague-Dawley rats were treated with the PCB mixture, Aroclor 1221 (A1221; 0, 0.1, 1, or 10 mg/kg), on Embryonic Days 16 and 18. Somatic and reproductive development of F1 and their F2 female offspring were monitored, including ages of eye opening, pubertal landmarks, and serum reproductive hormones. The results showed that low doses of A1221 given during this critical period of neuroendocrine development caused differential effects of A1221 on F1 and F2 female rats. In both generations, litter sex ratio was skewed toward females. In the F1 generation, additional effects were found, including a significant alteration of serum LH in the 1 mg/kg A1221 group. The F2 generation showed more profound alterations, particularly with respect to fluctuations in hormones and reproductive tract tissues across the estrous cycle. On proestrus, the day of the preovulatory GnRH/gonadotropin surge, F2 females whose mothers had been exposed perinatally to A1221 exhibited substantially suppressed LH and progesterone concentrations, and correspondingly smaller uterine and ovarian weights on estrus, compared with F2 descendants of control rats. These latter changes suggest a dysregulation of reproductive physiology. Thus, low levels of exposure to PCBs during late fetal development cause significant effects on the maturation and physiology of two generations of female offspring. These findings have implications for reproductive health and fertility of wildlife and humans.

NMDA receptor subunit NR2b: effects on LH release and GnRH gene expression in young and middle-aged female rats, with modulation by estradiol.

BACKGROUND/AIMS: The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. METHODS: Young (3-5 months) and middle-aged (10-13 months) female rats were ovariectomized (OVX), 17beta-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 min for 4 h, with ifenprodil (10 mg/kg) or vehicle injected (i.p.) after 1 h of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. RESULTS: Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. CONCLUSION: Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit play a role in GnRH-induced LH release, independent of de novo GnRH gene expression.

LHRH-(1-5): a bioactive peptide regulating reproduction.

Luteinizing hormone-releasing hormone-I (LHRH-I) was isolated from the mammalian hypothalamus and shown to be the primary regulator of reproduction through its initiation of pituitary gonadotropin release. Subsequently, it has also been shown to have non-pituitary actions. Although the regulation of LHRH-I synthesis and release has been extensively studied, there is additional evidence to suggest that processing of the peptide represents another layer of regulation. The focus of this review will be on evidence for the action of LHRH-(1-5), the pentapeptide metabolite of LHRH-I, in regulating LHRH-I synthesis, secretion and reproductive behavior. The involvement of LHRH-(1-5) in the control of aspects of reproduction might represent yet another level of regulatory complexity through neuropeptide processing.

Dissecting autocrine effects on pulsatile release of gonadotropin-releasing hormone in cultured rat hypothalamic tissue.

The control of reproductive function is manifested centrally through the control of hypothalamic release of gonadotropin-releasing hormone (GnRH) in episodic events or pulses. For GnRH release to occur in pulses, GnRH neurons must coordinate release events periodically to elicit a bolus of GnRH. We used a perifusion culture system to examine the release of GnRH from both intact hypothalami and enzymatically dispersed hypothalamic cells after challenge with GnRH analogs to evaluate the role of anatomical neuronal connections on autocrine/paracrine signals by GnRH on GnRH neurons. The potent GnRH agonist des-Gly(10)-D-Ala(6)-GnRH N-ethylamide, potent GnRH antagonists D-Phe(2)-D-Ala(6)-GnRH and D-Phe(2,6)-Pro(3)-GnRH or vehicle were infused, whereas GnRH release from hypothalamic tissue and cells were measured. PULSAR analysis of GnRH release profiles was conducted to evaluate parameters of pulsatile GnRH release. Infusion of the GnRH agonist resulted in a decrease in mean GnRH (P < 0.001), pulse nadir (P < 0.01), and pulse frequency (P < 0.05) but no effect on pulse amplitude. Infusion of GnRH antagonists resulted in an increase in mean GnRH (P < 0.001), pulse nadir (P < 0.05), and pulse frequency (P < 0.05) and in GnRH pulse amplitude only in dispersed cells (P < 0.05). These results are consistent with the hypothesis that GnRH inhibits endogenous GnRH release by an ultrashort-loop feedback mechanism and that treatment of hypothalamic tissue or cells with GnRH agonist inhibits ultrashort-loop feedback, whereas treatment with antagonists disrupts normal feedback to GnRH neurons and elicits an increased GnRH signal.

Aging-related changes in release of growth hormone and luteinizing hormone in female rhesus monkeys.

A decline in somatic function with aging in women is associated with a decrease in GH release and a loss of estrogen after menopause. As an initial step to establish a monkey model for the neuroendocrine mechanisms underlying somatopause and menopause, we have conducted three experiments in unrestrained aged (approximately 25.7-yr-old) and young (approximately 5.4-yr-old) female rhesus monkeys. GH release was pulsatile, and mean GH release and pulse amplitude were significantly lower in aged monkeys than in young monkeys. Injection of GHRH alone, GH-releasing peptide-2 alone, or the combination of both induced an increase in GH release in both age groups. The mean LH level, pulse amplitude, and baseline LH levels were significantly higher in aged animals than in young animals. Both estrogen and IGF-I levels were lower in aged than young monkeys. These results suggest that in female rhesus monkeys 1) there is a clear decline in circulating GH and IGF-I levels with aging; 2) GHRH and GH-releasing peptide-2 stimulate GH release synergistically; and 3) circulating LH levels increase as estrogen decreases with aging. These results indicate that the rhesus monkey is an excellent model for studies of the neuroendocrine mechanisms of aging.